Critical assessment of DNA adenine methylation in eukaryotes using quantitative deconvolution.

The discovery of N6-methyldeoxyadenine (6mA) across eukaryotes led to a search for additional epigenetic mechanisms. However, some studies have highlighted confounding factors that challenge the prevalence of 6mA in eukaryotes. We developed a metagenomic method to quantitatively deconvolve 6mA events from a genomic DNA sample into species of interest, genomic regions, and sources of contamination. Applying this method, we observed high-resolution 6mA deposition in two protozoa. We found that commensal or soil bacteria explained the vast majority of 6mA in insect and plant samples. We found no evidence of high abundance of 6mA in Drosophila, Arabidopsis, or humans. Plasmids used for genetic manipulation, even those from Dam methyltransferase mutant Escherichia coli, could carry abundant 6mA, confounding the evaluation of candidate 6mA methyltransferases and demethylases. On the basis of this work, we advocate for a reassessment of 6mA in eukaryotes.

Assessment of two DNA extraction kits for profiling poultry respiratory microbiota from multiple sample types.

Characterization of poultry microbiota is becoming increasingly important due to the growing need for microbiome-based interventions to improve poultry health and production performance. However, the lack of standardized protocols for sampling, sample processing, DNA extraction, sequencing, and bioinformatic analysis can hinder data comparison between studies. Here, we investigated how the DNA extraction process affects microbial community compositions and diversity metrics in different chicken respiratory sample types including choanal and tracheal swabs, nasal cavity and tracheal washes, and lower respiratory lavage. We did a side-by-side comparison of the performances of Qiagen DNeasy blood and tissue (BT) and ZymoBIOMICS DNA Miniprep (ZB) kits. In general, samples extracted with the BT kit yielded higher concentrations of total DNA while those extracted with the ZB kit contained higher numbers of bacterial 16S rRNA gene copies per unit volume. Therefore, the samples were normalized to equal amounts of 16S rRNA gene copies prior to sequencing. For each sample type, all predominant bacterial taxa detected in samples extracted with one kit were present in replicate samples extracted with the other kit and did not show significant differences at the class level. However, a few differentially abundant shared taxa were observed at family and genus levels. Furthermore, between-kit differences in alpha and beta diversity metrics at the amplicon sequence variant level were statistically indistinguishable. Therefore, both kits perform similarly in terms of 16S rRNA gene-based poultry microbiome analysis for the sample types analyzed in this study.

Low-Cost Quantitative Photothermal Genetic Detection of Pathogens on a Paper Hybrid Device Using a Thermometer.

Tuberculosis (TB), one of the deadliest infectious diseases, is caused by Mycobacterium tuberculosis (MTB) and remains a public health problem nowadays. Conventional MTB DNA detection methods require sophisticated infrastructure and well-trained personnel, which leads to increasing complexity and high cost for diagnostics and limits their wide accessibility in low-resource settings. To address these issues, we have developed a low-cost photothermal biosensing method for the quantitative genetic detection of pathogens such as MTB DNA on a paper hybrid device using a thermometer. First, DNA capture probes were simply immobilized on paper through a one-step surface modification process. After DNA sandwich hybridization, oligonucleotide-functionalized gold nanoparticles (AuNPs) were introduced on paper and then catalyzed the oxidation reaction of 3,3',5,5'-tetramethylbenzidine (TMB). The produced oxidized TMB, acting as a strong photothermal agent, was used for the photothermal biosensing of MTB DNA under 808 nm laser irradiation. Under optimal conditions, the on-chip quantitative detection of the target DNA was readily achieved using an inexpensive thermometer as a signal recorder. This method does not require any expensive analytical instrumentation but can achieve higher sensitivity and there are no color interference issues, compared to conventional colorimetric methods. The method was further validated by detecting genomic DNA with high specificity. To the best of our knowledge, this is the first photothermal biosensing strategy for quantitative nucleic acid analysis on microfluidics using a thermometer, which brings fresh inspirations on the development of simple, low-cost, and miniaturized photothermal diagnostic platforms for quantitative detection of a variety of diseases at the point of care.

Target search and recognition mechanisms of glycosylase AlkD revealed by scanning FRET-FCS and Markov state models.

DNA glycosylase is responsible for repairing DNA damage to maintain the genome stability and integrity. However, how glycosylase can efficiently and accurately recognize DNA lesions across the enormous DNA genome remains elusive. It has been hypothesized that glycosylase translocates along the DNA by alternating between a fast but low-accuracy diffusion mode and a slow but high-accuracy mode when searching for DNA lesions. However, the slow mode has not been successfully characterized due to the limitation in the spatial and temporal resolutions of current experimental techniques. Using a newly developed scanning fluorescence resonance energy transfer (FRET)-fluorescence correlation spectroscopy (FCS) platform, we were able to observe both slow and fast modes of glycosylase AlkD translocating on double-stranded DNA (dsDNA), reaching the temporal resolution of microsecond and spatial resolution of subnanometer. The underlying molecular mechanism of the slow mode was further elucidated by Markov state model built from extensive all-atom molecular dynamics simulations. We found that in the slow mode, AlkD follows an asymmetric diffusion pathway, i.e., rotation followed by translation. Furthermore, the essential role of Y27 in AlkD diffusion dynamics was identified both experimentally and computationally. Our results provided mechanistic insights on how conformational dynamics of AlkD-dsDNA complex coordinate different diffusion modes to accomplish the search for DNA lesions with high efficiency and accuracy. We anticipate that the mechanism adopted by AlkD to search for DNA lesions could be a general one utilized by other glycosylases and DNA binding proteins.

A lattice kinetic Monte-Carlo method for simulating chromosomal dynamics and other (non-)equilibrium bio-assemblies.

Biological assemblies in living cells such as chromosomes constitute large many-body systems that operate in a fluctuating, out-of-equilibrium environment. Since a brute-force simulation of that many degrees of freedom is currently computationally unfeasible, it is necessary to perform coarse-grained stochastic simulations. Here, we develop all tools necessary to write a lattice kinetic Monte-Carlo (LKMC) algorithm capable of performing such simulations. We discuss the validity and limits of this approach by testing the results of the simulation method in simple settings. Importantly, we illustrate how at large external forces Metropolis-Hastings kinetics violate the fluctuation-dissipation and steady-state fluctuation theorems and discuss better alternatives. Although this simulation framework is rather general, we demonstrate our approach using a DNA polymer with interacting SMC condensin loop-extruding enzymes. Specifically, we show that the scaling behavior of the loop-size distributions that we obtain in our LKMC simulations of this SMC-DNA system is consistent with that reported in other studies using Brownian dynamics simulations and analytic approaches. Moreover, we find that the irreversible dynamics of these enzymes under certain conditions result in frozen, sterically jammed polymer configurations, highlighting a potential pitfall of this approach.

A LAMP assay for the rapid and robust assessment of Wolbachia infection in Aedes aegypti under field and laboratory conditions.

With Wolbachia-based arbovirus control programs being scaled and operationalised around the world, cost effective and reliable detection of Wolbachia in field samples and laboratory stocks is essential for quality control. Here we validate a modified loop-mediated isothermal amplification (LAMP) assay for routine scoring of Wolbachia in mosquitoes from laboratory cultures and the field, applicable to any setting. We show that this assay is a rapid and robust method for highly sensitive and specific detection of wAlbB Wolbachia infection within Aedes aegypti under a variety of conditions. We test the quantitative nature of the assay by evaluating pooled mixtures of Wolbachia-infected and uninfected mosquitoes and show that it is capable of estimating infection frequencies, potentially circumventing the need to perform large-scale individual analysis for wAlbB infection status in the course of field monitoring. These results indicate that LAMP assays are useful for routine screening particularly under field conditions away from laboratory facilities.

Comparison of long-read sequencing technologies in the hybrid assembly of complex bacterial genomes.

Illumina sequencing allows rapid, cheap and accurate whole genome bacterial analyses, but short reads (<300 bp) do not usually enable complete genome assembly. Long-read sequencing greatly assists with resolving complex bacterial genomes, particularly when combined with short-read Illumina data (hybrid assembly). However, it is not clear how different long-read sequencing methods affect hybrid assembly accuracy. Relative automation of the assembly process is also crucial to facilitating high-throughput complete bacterial genome reconstruction, avoiding multiple bespoke filtering and data manipulation steps. In this study, we compared hybrid assemblies for 20 bacterial isolates, including two reference strains, using Illumina sequencing and long reads from either Oxford Nanopore Technologies (ONT) or SMRT Pacific Biosciences (PacBio) sequencing platforms. We chose isolates from the family Enterobacteriaceae, as these frequently have highly plastic, repetitive genetic structures, and complete genome reconstruction for these species is relevant for a precise understanding of the epidemiology of antimicrobial resistance. We de novo assembled genomes using the hybrid assembler Unicycler and compared different read processing strategies, as well as comparing to long-read-only assembly with Flye followed by short-read polishing with Pilon. Hybrid assembly with either PacBio or ONT reads facilitated high-quality genome reconstruction, and was superior to the long-read assembly and polishing approach evaluated with respect to accuracy and completeness. Combining ONT and Illumina reads fully resolved most genomes without additional manual steps, and at a lower consumables cost per isolate in our setting. Automated hybrid assembly is a powerful tool for complete and accurate bacterial genome assembly.

A comparison of Lyse-It to other cellular sample preparation, bacterial lysing, and DNA fragmentation technologies.

The ability for safe and rapid pathogenic sample transportation and subsequent detection is an increasing challenge throughout the world. Herein, we describe and use bead-beating, vortex, sonication, 903 protein saver cards, and Lyse-It methods, aiming to inactivate Gram-positive and -negative bacteria with subsequent genome DNA (quantitative Polymerase Chain Reaction) qPCR detection. The basic concepts behind the four chosen technologies is their versatility, cost, and ease of use in developed and underdeveloped countries. The four methods target the testing of bacterial resilience, cellular extraction from general and complex media and subsequent DNA extraction for qPCR detection and amplification. These results demonstrate that conventional high temperature heating, 903 protein saver cards, and Lyse-It are all viable options for inactivating bacterial growth for safe shipping. Additionally, Lyse-It was found to be particularly useful as this technology can inactivate bacteria, extract cells from 903 protein saver cards, lyse bacterial cells, and additionally keep genomic DNA viable for qPCR detection.

Rapid and sensitive detection of Anaplasma phagocytophilum using a newly developed recombinase polymerase amplification assay.

Anaplasma phagocytophilum, the bacterial pathogen responsible for tick-borne fever and human granulocytic anaplasmosis, can seriously affect the health of humans and a wide range of other mammals. In this study, we developed a recombinase polymerase amplification (RPA) assay to detect A. phagocytophilum in clinical samples. Following alignment of the relevant DNA sequences, a pair of specific primers based on the 16S rRNA gene was designed to specifically detect A. phagocytophilum. The assay was performed at a constant temperature of 38 °C for 30 min, with a final primer concentration of 0.4 µM. The specificity of the primers was confirmed when DNA from A. phagocytophilum was used as the positive control, and DNA from other related pathogens were used as the negative controls, with ddH2O acting as the blank control. The results showed that the primers did not cross-react with DNA from the other related pathogens. The assay's detection limit was 1.77 × 10-5 ng/µl, a 10 × higher sensitivity level than that determined for nested PCR. The RPA assay's performance was evaluated using 44 clinical samples, and the prevalence results for A. phagocytophilum were found to not differ significantly between the RPA assay and the nested PCR. Thus, we have developed a specific, sensitive, rapid and cost-effective RPA method, requiring only a water bath, for the detection of A. phagocytophilum. The assay should be especially useful in resource-limited areas where access to laboratory equipment is limited.

Bacterial chromosome organization. I. Crucial role of release of topological constraints and molecular crowders.

We showed in our previous studies that just 3% cross-links (CLs), at special points along the contour of the bacterial DNA, help the DNA-polymer to get organized at micron length scales [T. Agarwal et al., J. Phys.: Condens. Matter 30, 034003 (2018) and T. Agarwal et al., EPL (Europhys. Lett.) 121, 18004 (2018)]. In this work, we investigate how does the release of topological constraints help in the "organization" of the DNA-polymer. Furthermore, we show that the chain compaction induced by the crowded environment in the bacterial cytoplasm contributes to the organization of the DNA-polymer. We model the DNA chain as a flexible bead-spring ring polymer, where each bead represents 1000 base pairs. The specific positions of the CLs have been taken from the experimental contact maps of the bacteria Caulobacter crescentus and Escherichia coli. We introduce different extents of ease of release of topological constraints in our model by systematically changing the diameter of the monomer bead. It varies from the value where chain crossing can occur freely to the value where chain crossing is disallowed. We also study the role of compaction of the chain due to molecular crowders by introducing an "effective" weak Lennard-Jones attraction between the monomers. Using Monte Carlo simulations, we show that the release of topological constraints and the crowding environment play a crucial role to obtain a unique organization of the polymer.

Real-time PCR followed by high-resolution melting analysis - a new robust approach to evaluate SCCmec typing of methicillin-resistant Staphylococcus aureus.

AIM: We designed a novel approach based on real-time PCR followed by high-resolution melting (HRM) analysis to determine the Staphylococcal cassette chromosome mec (SCCmec) types of methicillin-resistant Staphylococcus aureus  strains, which we compared against the results of conventional multiplex PCR SCCmec typing. METHODS: Multiplex PCR (for ccr and mec gene complexes) was carried out as conventional method. The HRM analysis was then designed using standard strains of each SCCmec type. RESULTS: The M-PCR results included types III (33.33%), IV (43.33%) and V (23.33%). HRM analysis was able to distinguish all five types, which were used to set up the protocol with a sensitivity and specificity of 100% compared with the conventional method. CONCLUSION: This novel method for SCCmec typing has high specificity and sensitivity and can be conducted in a shorter period of time at lower costs.

Comparative assessment of qPCR enumeration methods that discriminate between live and dead Escherichia coli O157:H7 on beef.

Quantitative Polymerase Chain Reaction (qPCR) is a molecular method commonly used to detect and quantify bacterial DNA on food but is limited by its inability to distinguish between live and dead cell DNA. To overcome this obstacle, propidium monoazide (PMA) alone or with deoxycholate (DC) was used to prevent dead cell detection in qPCR. qPCR methods were used to detect strains of Escherichia coli O157, which can cause infection in humans with an infectious dose of less than 10 cells. A 5 strain E. coli O157:H7 cocktail was inoculated onto beef steaks and treated with interventions used in meat facilities (lactic acid (5%), peroxyacetic acid (200 ppm) or hot water (80 °C for 10 s)). Treatment of PMA or PMA + DC was applied to samples followed by DNA extraction and quantification in qPCR. RNA was also quantified in addition to conventional plating. For lactic acid intervention, qPCR DNA quantification of E. coli O157:H7 yielded 6.59 ±â€¯0.21 and 6.30 ±â€¯0.11 log gene copy #/cm2 for control and lactic acid samples, respectively and after treatment with PMA or PMA + DC this was further reduced to 6.31 ± 0.21 and 5.58 ± 0.38, respectively. This trend was also observed for peroxyacetic acid and hot water interventions. In comparison, RNA quantification yielded 7.65 ± 0.13 and 7.02 ± 0.38 log reverse transcript/cm2 for rRNA control and lactic acid samples, respectively, and for plating (LB), 7.51 ±â€¯0.06 and 6.86 ±â€¯0.32 log CFU/cm2, respectively. Our research determined that treatment of PMA + DC in conjunction with qPCR prevented dead cell DNA detection. However, it also killed cells injured from intervention that may have otherwise recovered. RNA quantification was more laborious and results had higher variability. Overall, quantification with conventional plating proved to be the most robust and reliable method for live EHEC detection on beef.

Assessment of pig saliva as a Streptococcus suis reservoir and potential source of infection on farms by use of a novel quantitative polymerase chain reaction assay.

OBJECTIVE To evaluate colonization of Streptococcus suis and Streptococcus parasuis on pig farms in Japan and to identify sources of infections. SAMPLE Saliva, feces, and vaginal swab samples from 84 healthy pigs of several growth stages on 4 farms and swab samples of feed troughs and water dispensers at those farms. PROCEDURES Samples were collected from August 2015 to June 2016. Two quantitative PCR (qPCR) assays (one for S suis and the other for S parasuis) were designed for use in the study. The novel qPCR assays were used in combination with previously described qPCR assays for S suis serotype 2 or 1/2 and total bacteria. Relative abundance of bacteria in each sample was evaluated. RESULTS Streptococcus suis was detected in all saliva samples and some of the other samples, whereas S parasuis was not detected in any of the samples, including saliva samples, which indicated a difference in colonization preference. The ratio of S suis to total bacteria in saliva appeared to increase with age of pigs. Streptococcus suis serotype 2 or 1/2 was detected in a few saliva samples and feed trough swab samples at 2 farms where S suis infections were prevalent. CONCLUSIONS AND CLINICAL RELEVANCE Saliva, especially that of sows, appeared to be a reservoir and source of S suis infection for pigs. The qPCR assay described here may provide an effective way to monitor for S suis in live pigs, which could lead to effective disease control on pig farms.

DNA sequence-based re-assessment of archived Cronobacter sakazakii strains isolated from dairy products imported into China between 2005 and 2006.

BACKGROUND: Cronobacter species are associated with severe foodborne infections in neonates and infants, with particular pathovars associated with specific clinical presentations. However, before 2008 the genus was regarded as a single species named Enterobacter sakazakii which was subdivided into 8 phenotypes. This study re-analyzed, using multi-locus sequence typing (MLST) and whole genome sequence with single nucleotide polymorphism analysis (WGS-SNP), 52 strains which had been identified as Enterobacter sakazakii as according to the convention at the time of isolation. These strains had been isolated from dairy product imports into China from 9 countries between 2005 and 6. Bioinformatic analysis was then used to analyze the relatedness and global dissemination of these strains. RESULT: FusA allele sequencing revealed that 49/52 strains were Cronobacter sakazakii, while the remaining 3 strains were Escherichia coli, Enterobacter cloacae, and Franconibacter helveticus. The C. sakazakii strains comprised of 8 sequence types (STs) which included the neonatal pathovars ST1, ST4 and ST12. The predominant sequence type was ST13 (65.3%, 32/49) which had been isolated from dairy products imported from 6 countries. WGS-SNP analysis of the 32 C. sakazakii ST13 strains revealed 5 clusters and 5 unique strains which did not correlate with the country of product origin. CONCLUSION: The mis-identification of E. coli, E. cloacae and F. helveticus as Cronobacter spp. reinforces the need to apply reliable methods to reduce the incidence of false positive and false negative results which may be of clinical significance. The WGS-SNP analysis demonstrated that indistinguishable Cronobacter strains within a sequence type can be unrelated, and may originate from multiple sources. The use of WGS-SNP analysis to distinguishing of strains within a sequence type has important relevance for tracing the source of outbreaks due to Cronobacter spp.

N6-Methylation Assessment in Escherichia coli 23S rRNA Utilizing a Bulge Loop in an RNA-DNA Hybrid.

We propose a sequence-selective assay of N6-methyl-adenosine (m6A) in RNA without PCR or reverse transcription, by employing a hybridization assay with a DNA probe designed to form a bulge loop at the position of a target modified nucleotide. The m6A in the bulge in the RNA-DNA hybrid was assumed to be sufficiently mobile to be selectively recognized by an anti-m6A antibody with a high affinity. By employing a surface-plasmon-resonance measurement or using a microtiter-plate immunoassay method, a specific m6A in the Escherichia coli 23S rRNA sequence could be detected at the nanomolar level when synthesized and purified oligo-RNA fragments were used for measurement. We have successfully achieved the first selective detection of m6A2030 specifically in 23S rRNA from real samples of E. coli total RNA by using our immunochemical approach.

Mechanistic DNA damage simulations in Geant4-DNA Part 2: Electron and proton damage in a bacterial cell.

We extended a generic Geant4 application for mechanistic DNA damage simulations to an Escherichia coli cell geometry, finding electron damage yields and proton damage yields largely in line with experimental results. Depending on the simulation of radical scavenging, electrons double strand breaks (DSBs) yields range from 0.004 to 0.010 DSB Gy-1 Mbp-1, while protons have yields ranging from 0.004 DSB Gy-1 Mbp-1 at low LETs and with strict assumptions concerning scavenging, up to 0.020 DSB Gy-1 Mbp-1 at high LETs and when scavenging is weakest. Mechanistic DNA damage simulations can provide important limits on the extent to which physical processes can impact biology in low background experiments. We demonstrate the utility of these studies for low dose radiation biology calculating that in E. coli, the median rate at which the radiation background induces double strand breaks is 2.8 × 10-8 DSB day-1, significantly less than the mutation rate per generation measured in E. coli, which is on the order of 10-3.

Role of special cross-links in structure formation of bacterial DNA polymer.

Using data from contact maps of the DNA-polymer of Escherichia coli (E. Coli) (at kilobase pair resolution) as an input to our model, we introduce cross-links between monomers in a bead-spring model of a ring polymer at very specific points along the chain. Via suitable Monte Carlo simulations, we show that the presence of these cross-links leads to a particular organization of the chain at large (micron) length scales of the DNA. We also investigate the structure of a ring polymer with an equal number of cross-links at random positions along the chain. We find that though the polymer does get organized at the large length scales, the nature of the organization is quite different from the organization observed with cross-links at specific biologically determined positions. We used the contact map of E. Coli bacteria which has around 4.6 million base pairs in a single circular chromosome. In our coarse-grained flexible ring polymer model, we used 4642 monomer beads and observed that around 80 cross-links are enough to induce the large-scale organization of the molecule accounting for statistical fluctuations caused by thermal energy. The length of a DNA chain even of a simple bacterial cell such as E. Coli is much longer than typical proteins, hence we avoided methods used to tackle protein folding problems. We define new suitable quantities to identify the large scale structure of a polymer chain with a few cross-links.

Assessment of variation in microbial community amplicon sequencing by the Microbiome Quality Control (MBQC) project consortium.

In order for human microbiome studies to translate into actionable outcomes for health, meta-analysis of reproducible data from population-scale cohorts is needed. Achieving sufficient reproducibility in microbiome research has proven challenging. We report a baseline investigation of variability in taxonomic profiling for the Microbiome Quality Control (MBQC) project baseline study (MBQC-base). Blinded specimen sets from human stool, chemostats, and artificial microbial communities were sequenced by 15 laboratories and analyzed using nine bioinformatics protocols. Variability depended most on biospecimen type and origin, followed by DNA extraction, sample handling environment, and bioinformatics. Analysis of artificial community specimens revealed differences in extraction efficiency and bioinformatic classification. These results may guide researchers in experimental design choices for gut microbiome studies.

An Affordable Microsphere-Based Device for Visual Assessment of Water Quality.

This work developed a prototype of an affordable, long-term water quality detection device that provides a visual readout upon detecting bacterial contamination. This device prototype consists of: (1) enzyme-releasing microspheres that lyse bacteria present in a sample, (2) microspheres that release probes that bind the DNA of the lysed bacteria, and (3) a detector region consisting of gold nanoparticles. The probes bind bacterial DNA, forming complexes. These complexes induce aggregation of the gold nanoparticles located in the detector region. The nanoparticle aggregation process causes a red to blue color change, providing a visual indicator of contamination being detected. Our group fabricated and characterized microspheres made of poly (ε-caprolactone) that released lysozyme (an enzyme that degrades bacterial cell walls) and hairpin DNA probes that bind to regions of the Escherichiacoli genome over a 28-day time course. The released lysozyme retained its ability to lyse bacteria. We then showed that combining these components with gold nanoparticles followed by exposure to an E. coli-contaminated water sample (concentrations tested-106 and 108 cells/mL) resulted in a dramatic red to blue color change. Overall, this device represents a novel low-cost system for long term detection of bacteria in a water supply and other applications.

Modeling Bacterial DNA: Simulation of Self-Avoiding Supercoiled Worm-Like Chains Including Structural Transitions of the Helix.

Under supercoiling constraints, naked DNA, such as a large part of bacterial DNA, folds into braided structures called plectonemes. The double-helix can also undergo local structural transitions, leading to the formation of denaturation bubbles and other alternative structures. Various polymer models have been developed to capture these properties, with Monte-Carlo (MC) approaches dedicated to the inference of thermodynamic properties. In this chapter, we explain how to perform such Monte-Carlo simulations, following two objectives. On one hand, we present the self-avoiding supercoiled Worm-Like Chain (ssWLC) model, which is known to capture the folding properties of supercoiled DNA, and provide a detailed explanation of a standard MC simulation method. On the other hand, we explain how to extend this ssWLC model to include structural transitions of the helix.